Role of Tet proteins in epigenetic regulation of embryonic stem cell biology

修改项目摘要/摘要部分： 本项目的主要目标是剖析泰特酶在胚胎干细胞中的基因调控功能，并确定这些蛋白质在基因抑制和干细胞生物学中的非经典（非酶）作用的意义。十-十一易位（泰特）酶（Tet 1、Tet 2、Tet 3）调节ESCs中和发育过程中的基因表达程序。它们通过将5-甲基胞嘧啶（5 mC）氧化为5-羟甲基胞嘧啶和其他衍生物来促进DNA去甲基化。泰特酶在DNA去甲基化中的酶功能及其在ESCs和发育中的意义已经作为我在该领域先前工作的一部分进行了很好的研究。然而，我的实验室最近的发现强烈表明，泰特蛋白的关键生物学作用与其催化活性无关。我们已经发现，更多的基因差异表达Tet 1敲除胚胎干细胞比Tet 1催化突变型胚胎干细胞（与野生型胚胎干细胞）。这些基因中的大多数是过表达的，这表明泰特酶的非催化功能调节ESCs中的基因阻遏，并有助于其生物学。因此，我们发现无催化活性的泰特酶拯救了泰特缺陷型ESC的一些表型。这些观察为这个项目奠定了初步的前提。我们推测，除了它们的DNA脱甲基酶活性，泰特酶调节表观基因组独立于它们的酶活性。这涉及将染色质阻遏复合物募集到基因调控区以进行适当的基因阻遏。这构成了一个新的表观遗传调控层，影响ESC生物学的关键特征，包括增殖以及体外分化和谱系特化。利用我实验室最近开发的由泰特催化突变体和敲除ESC组成的平台，我们将应用分子、细胞和发育方法来剖析干细胞中泰特酶的催化依赖性和非催化性需求，并建立泰特酶在ESC生物学中的非经典作用的意义。 我们提出以下目标：（1）我们将确定泰特酶在将染色质阻遏复合物Sin 3a和PRC 2募集到基因沉默的基因调控元件中的非经典作用。(2)我们将定义Tet-mediated非经典基因抑制在ESC增殖，以及在体外内胚层和滋养外胚层分化和谱系规范的生物学相关性。我们的研究结果将揭示泰特酶调控胚胎干细胞的新的表观遗传机制。这将增强我们对干细胞中表观遗传网络的理解，并将为研究发育和疾病过程中Tet-mediated基因调控开辟未来的机会。

Modified Project Summary/Abstract Section: The main goal of this project is to dissect gene regulatory functions of Tet enzymes in ESCs, and define the significance of noncanonical (non-enzymatic) roles of these proteins in gene repression and in stem cell biology. The Ten-eleven-translocation (Tet) enzymes (Tet1, Tet2, Tet3) regulate gene expression program in ESCs and during development. They facilitate DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5- hydroxymethylcytosine and other derivatives. The enzymatic function of Tet enzymes in DNA demethylation and its significance in ESCs and development has been well investigated as part of my prior work in the field. However, recent findings from my lab strongly suggest that key biological roles of Tet proteins are independent of their catalytic activity. We have found that more genes are differentially expressed in Tet1 knockout ESCs than in Tet1 catalytic mutant ESCs (vs. wild type ESCs). Majority of these genes are overexpressed, suggesting that noncatalytic functions of Tet enzymes regulate gene repression in ESCs and contribute to their biology. Consistently, we found that catalytically inactive Tet enzymes rescued some of the phenotypes of Tet deficient ESCs. These observations build the initial premise for this project. We hypothesize that in addition to their DNA demethylase activity, Tet enzymes regulate the epigenome independent of their enzymatic activity. This involves recruiting chromatin repressive complexes to gene regulatory regions for proper gene repression. This constitutes a novel layer of epigenetic regulation that influences key features of ESC biology including proliferation as well as in vitro differentiation and lineage specification. Using a recently developed platform in my lab that consists of Tet catalytic mutant and knockout ESCs, we will apply molecular, cellular and developmental approaches to dissect the catalytic dependent and independent requirements of Tet enzymes in stem cells, and establish the significance of noncanonical roles of Tet enzymes in ESC biology. We propose the following aims: (1) We will establish the noncanonical roles of Tet enzymes in recruiting chromatin repressive complexes, Sin3a and PRC2, to gene regulatory elements for gene silencing. (2) We will define the biological relevance of Tet-mediated noncanonical gene repression in ESC proliferation, as well as in in vitro endodermal and trophectodermal differentiation and lineage specification. Our findings will reveal novel epigenetic mechanisms of regulation of ESCs by Tet enzymes. This will enhance our understanding of the epigenetic networks in stem cells and will open future opportunities for investigating Tet-mediated gene regulation during development and in diseases.